Method of determining thiopurine methyltransferase activity

ABSTRACT

The present invention provides a method of determining thiopurine methyltransferase (TPMT) activity in a subject. The method includes the steps of reacting sample obtained from the subject with a thiopurine derivative that is not 6-mercaptopurine to produce a methylated purine product; contacting the reacted sample with acid, thereby precipitating proteinaceous material from the reacted sample; separating supernatant from the precipitated proteinaceous material; and detecting in the supernatant the methylated purine product, where the amount of the methylated purine product indicates a level of thiopurine methyltransferase activity in the subject. In a method of the invention, the subject can be, for example, an inflammatory bowel disease patient. In one embodiment, the acid used to precipitate proteinaceous material is perchloric acid, for example, 70% perchloric acid. In another embodiment, the thiopurine derivative used as a substrate is 6-thioguanine. In a further embodiment, the methylated purine product is detected by fluorescence, which can be combined, if desired, with high performance liquid chromatography (HPLC).

This application is based on, and claims the benefit of, U.S.Provisional Application No. 60/205,695, filed May 19, 2000, and entitledMethod of Determining Thiopurine Methyltransferase Activity, and whichis incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to thiopurine drugs used for thetreatment of inflammatory bowel disease, leukemia, and organtransplantation rejection, and more specifically to methods fordetermining thiopurine methyltransferase activity in order toindividualize dosages of 6-mercaptopurine therapy.

2. Background Information

Mercaptopurine (6-MP or 6-thiopurine) and azathioprine[6-(1-methyl-4-nitro-5-imidazolylthio)purine] are cytotoxic drugs whichare effective in the treatment of ulcerative colitis and Crohn's disease(Present et al., Annals of Internal Medicine 111:641-649 (1989)). Bothare immunosuppressive agents that act as purine antagonists and therebyinhibit the synthesis of DNA, RNA and proteins (Lennard, EuropeanJournal of Clinical Pharmacology 43:329-339 (1992)). 6-MP was initiallyused for the treatment of childhood acute lymphoblastic leukemia and forpost-operative treatment of organ transplantation surgery (Burchenal etal., Blood 8:965-999 (1953)), and its use has since been extended torheumatoid arthritis and inflammatory bowel disease (Kirschner,Gastroenterology 115:813-821 (1998)).

The prodrug azathioprine (AZA) is rapidly converted to 6-mercaptopurinethrough non-enzymatic, nucleophilic attack by sulfhydryl-containingcompounds in the circulation. 6-MP and azathioprine (AZA), which areforms of the same drug and metabolic precursors of the activecomponents, are acted upon by at least three competing enzymaticpathways (Lennard, supra, 1992). An overview of the action of theseenzymes is shown in FIG. 1. As shown in FIG. 2, several major enzymepathways are involved. Xanthine oxidase (XO) converts 6-mercaptopurineto 6-thiouric acid. Hypoxanthine phosphoribosyl transferase (HPRT)converts 6-mercaptopurine to 6-thioinosine-5′-monophosphate, which is aprecursor to 6-thioguanine nucleotides. Thiopurine methyltransferase(TPMT) catalyzes the S-methylation of 6-mercaptopurine tomethylmercaptopurine (6-MMP). Thus, 6-mercaptopurine is enzymaticallyconverted to various metabolites, including 6-thioguanine (6-TG) and6-thioguanine nucleotides, which are the presumptive active metabolitesmediating the effects of azathioprine/6-mercaptopurine drug therapy.

The interplay of the pathways described above is genetically determinedand creates a highly individualized response toazathioprine/6-mercaptopurine drug therapy. The population frequencydistribution of TPMT enzyme is trimodal, with the majority ofindividuals (89%) having high activity, 11% having intermediate activityand about 1 in 300 (0.33%) having undetectable activity (Weinshilboumand Sladek, Amer. J. Human Genetics 32:651-662 (1980)). Such a trimodalrelationship has been confirmed by direct measurements of TPMT enzymeactivity by the Kröplin HPLC assay method (Kroplin et al., Eur. J. Clin.Pharmacol., 54 265-271 (1998)). In contrast to variation in TPMTactivity, there is very little inter-individual variation in XO activityand only limited data on HPRT activity (Lennard, Eur. J. Clin. Pharm.,43:329-339 (1992)).

Available evidence indicates that TPMT activity effectively modulatesthe concentration of 6-thioguanine by shunting 6-mercaptopurine into theproduction of 6-methyl-mercaptopurine. Patients who less efficientlymethylate these thiopurines have more extensive conversion to6-thioguanine nucleotides, which can lead to potentially fatalhematopoietic toxicity. Thus, patients with intermediate or low TPMTactivity can be more susceptible to toxic side effects ofazathioprine/6-mercaptopurine therapy (Present et al., Annals ofInternal Medicine 111:641-649 (1989)). Such toxic side effects includeallergic reactions, neoplasia, opportunistic infections, hepatitis, bonemarrow suppression, and pancreatitis; in about 1 out of 300 patients,this therapy cannot be tolerated. As a consequence, many physicians arereluctant to treat patients with azathioprine/6-mercaptopurine therapy,particularly due to the risk of infection and neoplasia.

Thus, there is a need for a method of optimizing the dose of6-mercaptopurine by determining the level of thiopurinemethyltransferase activity in a patient. Such a method would be valuablefor optimizing therapeutic efficacy of azathioprine/6-mercaptopurinetherapy while minimizing undesirable side effects. The present methodsatisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides a method of determining thiopurinemethyltransferase (TPMT) activity in a subject. The method includes thesteps of reacting sample obtained from the subject with a thiopurinederivative that is not 6-mercaptopurine to produce a methylated purineproduct; contacting the reacted sample with acid, thereby precipitatingproteinaceous material from the reacted sample; separating supernatantfrom the precipitated proteinaceous material; and detecting in thesupernatant the methylated purine product, where the amount of themethylated purine product indicates a level of thiopurinemethyltransferase activity in the subject. In a method of the invention,the subject can be, for example, an inflammatory bowel disease patient.In one embodiment, the acid used to precipitate proteinaceous materialis perchloric acid, for example, 70% perchloric acid. In anotherembodiment, the thiopurine derivative used as a substrate is6-thioguanine. In a further embodiment, the methylated purine product isdetected by fluorescence, which can be combined, if desired, with highperformance liquid chromatography (HPLC).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a general schematic of the metabolism of azathioprine and6-mercaptopurine. Oral azathioprine is rapidly converted to6-mercaptopurine by a nonenzymatic process. Initial 6-MP transformationsoccur along competing catabolic (XO, xanthine oxidase; TPMT) andanabolic (HPRT, hypoxanthine phosphoribosyltransferase) enzymaticpathways. Once formed by HPRT, 6-TIMP may be transformed into 6-TGN bythe rate-limiting enzyme inosine monophosphate dehydrogenase (IMPDH) ormethylated into 6-MMPR (Dubinsky et al., Gastroenterology 118:705-713(2000)).

FIG. 2 shows the metabolism of azathioprine and 6-mercaptopurine.6-mercaptopurine metabolic pathways are indicated by solid arrows;dashed arrows indicate putative products of dephosphorylation tonucleotides and further catabolism to nucleobases. HPRT, hypoxanthinephosphoribosyltransferase; TMPT, thiopurine methyltransferase; XO,xanthine oxidase; IMPD, inosine monophosphate dehydrogenase; GMPS,guanosine monophosphate synthetase.

DETAILED DESCRIPTION OF THE INVENTION

Effective treatment of inflammatory bowel diseases such as Crohn'sdisease and ulcerative colitis with 6-mercaptopurine and the relatedpro-drug, azathioprine (AZA), is complicated by the toxic side effectsof this therapy in patients with low or intermediate TPMT enzymeactivity. Such toxic side effects include allergic reactions, neoplasia,opportunistic infections, hepatitis, bone marrow suppression, andpancreatitis. Therefore, it is critical that TPMT activity be measuredin candidates for 6-mercaptopurine treatment in order to administer theappropriate dose to patients with intermediate levels of activity and toavoid therapy in patients with extremely low TMPT activity.Additionally, TPMT activity can be assayed in patients who have begunazathioprine/6-mercaptopurine therapy in order to ensure thatappropriate enzyme levels are present for the dose of drug administered.

The present invention is directed to the discovery of a rapid andreliable method for determining thiopurine methyltransferase (TPMT)activity in a subject and is particularly advantageous in that enzymeactivity can be determined even if the patient has already beenmedicated with 6-mercaptopurine or azathioprine. A method of theinvention for determining TPMT activity in a subject includes the stepsof reacting sample obtained from the subject with a thiopurinederivative that is not 6-mercaptopurine to produce a methylated purineproduct; contacting the reacted sample with acid, thereby precipitatingproteinaceous material from the reacted sample; separating supernatantfrom the precipitated proteinaceous material; and detecting in thesupernatant the methylated purine product, where the amount of themethylated purine product indicates a level of thiopurinemethyltransferase activity in the subject. In a method of the invention,the subject can be, for example, an inflammatory bowel disease patient.In one embodiment, the acid used to precipitate proteinaceous materialis perchloric acid, for example, 70% perchloric acid. In anotherembodiment, the thiopurine derivative used as a substrate is6-thioguanine. In a further embodiment, the methylated purine product isdetected by fluorescence, which can be combined, if desired, with highperformance liquid chromatography (HPLC).

Thus, the present invention is directed to a method for quantitativelymeasuring and monitoring the activity of TPMT in human peripheral blood.As disclosed in Example I, direct enzymatic turnover of a thiopurine(6-thioguanine) yielded the fluorescent product 6-methylthioguanine. Thelevel of the specific 6-methylthioguanine product resulting from theenzyme turnover was separated from other assay components by perchloricacid precipitation, followed by high performance liquid chromatography(HPLC) and quantitative detection by a high sensitivity fluorometricdetector. This novel assay method allows determination of TPMT enzymeactivity even if the patient is taking 6-MP or AZA therapy andstreamlines the sample extraction for rapid quantitation.

Using this procedure, patients were readily separated into three groups:those who were genetically wild type had enzyme levels of greater than23.60 nmoles 6-mTGN/g Hb/hr; those heterozygous for one of threepreviously characterized TPMT mutations had TMPT activity levels from6.76 to 23.6 nmoles 6-mTGN/g Hb/hour; and an individual homozygous lowat the TPMT locus had a TPMT activity level less than 6.76 6-mTGN/gHb/hour (1.1 6-mTGN/g Hb/hour). Thus, the level of fluorescentmethylated 6-thioguanine product (6-mTG) was correlated with TMPTactivity and can be used to prevent toxic side effects in patients withlow or intermediate TPMT activity levels.

The methods of the invention are particularly useful for treating IBD,or subtypes of IBD, which has been classified into the broad categoriesof Crohn's disease and ulcerative colitis. Crohn's disease (regionalenteritis) is a disease of chronic inflammation that can involve anypart of the gastrointestinal tract. Commonly, the distal portion of thesmall intestine (ileum) and cecum are affected. In other cases, thedisease is confined to the small intestine, colon or anorectal region.Crohn's disease occasionally involves the duodenum and stomach, and morerarely the esophagus and oral cavity. The most frequent symptoms ofCrohn's disease are abdominal pain, diarrhea and recurrent fever, andthis disease also can be associated with intestinal obstruction orfistula, which is an abnormal passage between diseased loops of bowel.Crohn's disease further can be associated with complications such asinflammation of the eye, joints and skin; liver disease; kidney stonesor amyloidosis.

The pathology of Crohn's disease includes transmural inflammation,involving all layers of the bowel wall. Thickening and edema, forexample, typically appear throughout the bowel wall, with fibrosis alsopresent in long-standing disease. Furthermore, the inflammationcharacteristic of Crohn's disease also is discontinuous in that segmentsof inflamed tissue, known as “skip lesions,” are separated by apparentlynormal intestine. Linear ulcerations, edema, and inflammation of theintervening tissue lead to a “cobblestone” appearance of the intestinalmucosa, which is distinctive of CD. A hallmark of Crohn's disease is thepresence of discrete aggregations of inflammatory cells, known asgranulomas, which are generally found in the submucosa (Rubin andFarber, Pathology (Second Edition) Philadelphia: J. B. LippincottCompany (1994)).

The inflammatory bowel disease ulcerative colitis (UC) is a disease ofthe large intestine characterized by chronic diarrhea with crampingabdominal pain, rectal bleeding, and loose discharges of blood, pus andmucus. The manifestations of ulcerative colitis vary widely. A patternof exacerbations and remissions typifies the clinical course of most UCpatients (70%), although continuous symptoms without remission arepresent in some patients with ulcerative colitis. Local and systemiccomplications of UC include arthritis, eye inflammation such as uveitis,skin ulcers and liver disease. In addition, ulcerative colitis, andespecially long-standing, extensive disease, is associated with anincreased risk of colon carcinoma.

Ulcerative colitis generally is a diffuse disease that usually extendsfrom the most distal part of the rectum for a variable distanceproximally. Sparing of the rectum or involvement of the right side(proximal portion) of the colon alone is unusual in ulcerative colitis.The inflammatory process of ulcerative colitis is limited to the colonand is distinguished by a superficial inflammation of the mucosa thatgenerally spares the deeper layers of the bowel wall. (Rubin and Farber,supra, 1994).

The methods of the invention are useful for determining TPMT activity ina variety of subjects, including patients having inflammatory boweldisease or leukemia, and organ or allograft transplant recipient. Asused herein, the term “subject” means any animal which expresses theenzyme thiopurine methyltransferase, including a human, non-humanprimate, rabbit, rat or mouse, and especially a human. A subject can bea patient having an inflammatory bowel disease (ulcerative colitis orCrohn's disease); leukemia; or a transplant recipient. A subject may ormay not have been treated with 6-mercaptopurine or azathioprine therapy.

As used herein, the term “sample” refers to biological material obtainedfrom a subject and encompasses any material that contains thecytoplasmic enzyme thiopurine methyltransferase. A sample can be, forexample, whole blood, plasma, saliva or other bodily fluid or tissuethat contains cells. A preferred sample is whole blood, from which redblood cells can be obtained as described in Example I.

The methods of the invention involve reacting a sample with a thiopurinederivative that is not 6-mercaptopurine. As used herein, the term“thiopurine derivative” refers to a thioxopurine, thiopurine or aderivative or analog thereof which acts as a methyl acceptor when actedupon by thiopurine methyltransferase. A thiopurine derivative useful inthe invention can be a naturally occurring or non-naturally occurringsubstrate for thiopurine methyltransferase which has a structure similarto a thiopurine or thioxopurine, for example, 6-thioguanine, and canoccur in reduced or oxidized form. Additional thiopurine derivatives areknown in the art and include thioinosine, thioadenine, thioguanosine andthioadenosine. It should be understood that any thiopurine derivativeother than 6-mercaptopurine can be useful in the methods of theinvention. Such a thiopurine derivative has the following structure

and is a fused 6.5 membered ring derivative with a thioxo or thio at the6 position of the 6-membered ring, ring nitrogens at the 1 and 3positions of the 6-membered ring and ring nitrogens at the 7 and 9positions of the 5-membered ring. For example, where R at the 2-positionof the 6-membered ring is amino, the thiopurine derivative isthioguanine. It is understood that the R groups at the 1, 2, 3, and 7,8, and 9 positions are independent and can be, for example, a sugar,halide, C1-C₆ alkyl, OH, amino, mono or di-substituted amino (wheresubstitutions are C₁-C₆ alkyl or substituted alkyl) or substituted C₁-C₆alkyl (where substitutions are one or more OH, halide, amino or mono ordisubstituted amino), provided that the R group does not inhibitthiopurine methyltransferase activity.

In one embodiment, a thiopurine derivative useful in the invention is acompound which fluoresces when methylated. For example, 6-thioguanine ismethylated by thiopurine methyltransferase to the fluorescent product6-methyl thioguanine. The skilled person understands that these andother non-6-MP substrates for thiopurine methyltransferase can bethiopurine derivatives useful in the invention.

As used herein, the term “methylated purine product” refers to thechemical product generated as a result of thiopurine methyltransferaseactivity upon a thiopurine derivative. Methylated purine productsinclude 6-methylthioguanine, and analogs thereof, which are produced bymethylation of 6-thioguanine.

In a method of the invention, proteinaceous material is precipitatedaway from the methylated purine product. As used herein, the term “acid”refers to a reagent that is capable of effecting preferentialprecipitation of proteinaceous material from solution, while methylatedpurine products such as 6-methylthioguanine are not precipitated. Oneskilled in the art understands that an acid useful in the invention doesnot substantially destroy, degrade or otherwise affect detection ofmethylated purine product. Exemplary acids useful in the invention aredisclosed herein as perchloric acid; sulfuric acid, phosphoric acid andglacial acetic acid (see Example III). Additional acids useful in theinvention can be identified by the ability to yield a substantiallysimilar TPMT activity level for a particular sample, as compared to asample contacted with 70% perchloric acid.

As used herein, the term “detecting” refers to a process by which theamount of methylated purine product is determined. Methylated purineproducts, such as 6-mTG, can be detected by a variety of quantitativetechniques suitable for distinguishing methylated purine products fromthiopurine derivatives and other compounds. Methylated purine productscan be conveniently detected, for example, using high performance liquidchromatography (HPLC). Additional methods for detecting methylatedpurine products include capillary electrophoresis, gas chromatography(GC), gas chromatography-mass spectroscopy (GC-MS) and thin layerchromatography (TLC). The skilled person understands that these andother methods for quantitating the amount of methylated purine productcan be useful in the invention.

The following examples are intended to illustrate but not limit thepresent invention.

EXAMPLE I Quantitative Determination of Thiopurine MethyltransferaseEnzyme Activity Levels in Human Red Blood Cells

This example describes quantitative determination of thiopurinemethyltransferase (TPMT) activity using 6-thioguanine as a substrate.

A. Blood Sample Preparation

Whole blood samples were washed in normal saline, and the hemoglobincount determined prior to freezing. Briefly, blood samples werecollected in ethylene diamine tetraacetic acid (EDTA), and theEDTA-treated whole blood centrifuged for 3 minutes at 3500 rpm at 10° C.in a tabletop centrifuge (Beckman, Fullerton, Calif.; Model #TJ-6R).Plasma was examined for hemolysis, and sample was rejected if hemolysiswas detected. After removing the plasma, packed cells were washed onetime with an equal volume of 0.9% saline and centrifuged as describedabove. A hemoglobin count was determined using a Coulter ONYX cellcounter. After counting, the washed packed red blood cells (RBCs) werediluted 1:5 with 0.02 M phosphate buffer (pH 7.4) and stored at −70° C.until analysis.

B. Enzymatic Reaction and Protein Precipitation

Red blood cell lysates were incubated with 6-thioguanine, andproteinaceous material subsequently precipitated with perchloric acid asdescribed below.

“Unknown” refers to an enzymatic reaction performed with a patientsample. “Baseline” refers to an enzymatic reaction performed with apatient sample without the methyl donor, S-adenosyl methionine (SAM).“Blank” refers to an enzymatic reaction performed with normal pooledcells without SAM. “Control” refers to an enzymatic reaction performedwith lysate having a previously determined “low” activity, “medium”activity, or “high” activity. Calibration standards refer to a6-methylthioguanine standard containing lysate but not SAM.

The following components were combined: 150 μL 3 mM 6-thioguanine in 0.1M Phosphate Buffer, pH 7.4 (Sigma Chemical, Catalog # A-4882); 50 μLdistilled H₂O for a “blank” or “baseline” sample or 0.16 mM SAM Solution(Sigma Chemical, St. Louis, Mo.; Catalog # A-7007) for an “unknown”sample; 50 μl thawed red blood cell lysate (patient cell lysate for“unknown” or “baseline”, normal pooled cell lysate for calibrationstandard and “blank” sample, control cell lysate for “control” sample);and 50 μl of 6-methylthioguanine standard for calibration standardsample.

Mixtures were incubated in a 37° C. shaking water bath for 1 hour andsubsequently cooled on ice for 5 to 60 minutes. A volume of 50 μL 70%perchloric acid (Fisher, Pittsburgh, Pa.; Catalog # MK2766-500) wasadded to a final concentration of 11.67%, and the acidified samplecentrifuged for 5 minutes to precipitate proteinaceous material.Supernatant (280 μL) was transferred to an HPLC autosampler vial(Hewlett-Packard, Palo Alto, Calif.; catalog # 5183-4504) fitted with aninsert (Hewlett-Packard, catalog # 5181-3377).

C. HPLC Detection of 6-methylthioguanine

HPLC with fluorescent detection was performed to measure theconcentration of 6-methylthioguanine in patient samples essentially asfollows.

A Hewlett Packard HPLC System (Model # 1100) with in-line fluorometer(Series 1100) was used for analysis. A C18 reverse phase column (Waters,Milford, Mass.; Part # 186000494) with an in-line filter(Hewlett-Packard, Catalog # 01090-68072) was equilibrated in freshlyprepared mobile phase (0.1 M H₃PO₄, 1 mM DTT in distilledH₂O/acetonitrile, 95/5%, v/v). A primer solution injection was includedin addition to calibration standards, controls and patient samples, andmatrix blanks were run immediately before and after the study or controlsamples.

Each sample (100 μL) was injected onto the column with a flow rate of1.2 mL/min and run under isocratic conditions. Fluorescence wasmonitored by setting the excitation wavelength to 315 nm and theemission wavelength to 390 nm. The retention time for6-methylthioguanine was 5.2 minutes ±20%.

D. Determination of TPMT Activity

Enzyme activity was expressed as nmol 6-methylthioguanine/gramhemoglobin in 50 μL of lysate/hour. Patient samples that had less than6.76 nmoles 6-methylthioguanine/g Hb/hour indicate a subject possessinglow TPMT activity. Patient samples having 6.76 to 23.60 nmoles6-methylthioguanine/g Hb/hour indicate a subject possessing intermediate(heterozygous) TPMT activity. Patient samples having greater than 23.60nmoles 6-methylthioguanine/g Hb/hour indicate a subject possessing high(wild type) TPMT activity.

EXAMPLE II Correlation of TPMT Activity with TPMT Genotype

This example demonstrates that the methods of the invention fordetermining TPMT activity produce results that correlate with TPMTgenotyping.

Previous studies of various ethnic populations have shown that roughly89% of individuals express wild type (high) TPMT activity, while about11% express intermediate activity and less than 1% (approximately 0.3%)express very low or undetectable TPMT activity. TPMT activity levelsassayed using perchloric acid precipitation followed by HPLC andfluorescence detection as described in Example I were correlated toseveral TPMT genotype classes: TPMT*2-G238C in exon 5; TPMT*3A-G460A inexon 7, and TPMT*3-A719G in exon 10. To a large degree, these threemutations determine TPMT enzyme activity levels in humans.

TPMT enzyme units and TPMT genotypes were measured for 44 individualswith a wild type genotype, 6 heterozygous individuals, and 1 homozygotelow individual, with red cell lysates coded and assayed in duplicate.The corresponding DNA was also coded and genotype was determined byProPredictR_(x) TPMT Genetic Analysis at Prometheus Laboratories (SanDiego, Calif.). Briefly, genotypes were determined by isolation andpurification of DNA from whole blood, following by amplification ofregions of interest by the polymerase chain reaction in the presence offluorescent-labeled primers. The three amplified regions on chromosome 6were the following: TPMT*2-G238C in exon 5, TPMT*3A-G460A in exon 7, andTPMT*3-A719G in exon 10. Subsequently, the labeled PCR products weredigested with specific restriction enzymes, and the digested and labeledPCR fragment products analyzed on an ABI Prism 310 Genetic Analyzerusing capillary electrophoresis. The mean, standard deviation, range ofresults, and 95% confidence interval were calculated for each genotypegroup. ANOVA was performed on the three genotype groups to determine thep values (significance) of the means between the groups.

The results of this analysis demonstrated that there was very gooddiscrimination of homozygote low patients as compared to heterozygoteand wild type patients (see Table 1). The distinction between thepatient which was homozygous low for TPMT agreed with a published reportin which all homozygote low patients had TPMT values below 2 nmoles6-mTGN/gm Hb/hour. The heterozygote group had TPMT values ranging from alow of 9.7 to a high of 19.8 nmoles 6-mTGN/gm Hb/hour, agreeing wellwith a published report in which heterozygotes had TPMT activitiesranging from a low of about 10 to an assigned high value of 23.5 nmoles6-mTGN/gm Hb/hour. One wild type TPMT activity level (19.6) overlappedwith the heterozygote range of 9.7-19.8 nmoles 6-mTGN/gm Hb/hour. Thisparticular patient was also measured by the Mayo Clinic radiochemicalassay and has been shown to have a somewhat low wild type phenotype(Mayo reported a value of 16.7 U/ml RBC as compared to their normalrange of 13.8-25.1 U/ml RBC).

The difference in mean TPMT activity between the homozygote low andheterozygote populations [X_(L-L)=1.1 versus X_(H-L)=15.18] shows thathomozygote low individuals can be reliably distinguished from thoseheterozygous for wild type TMPT and from those who are homozygous wildtype at the three TPMT polymorphic sites assayed (see Table 1).Furthermore, the unpaired t test indicates that the wild type andheterozygote populations were significantly different (p<0.0001,two-tailed). Due to the small sample size in the homozygote low group,which represents a rare genotype, a similar analysis could not beperformed between the wild type and homozygote low or heterozygote andhomozygote low groups.

TABLE 1 Results of Genotyping versus Phenotyping Correlation for TPMTHomo- Homo- Wild Wild Hetero- Hetero- zygote zygote Parameter: Type Typezygote zygote Low Low Study: This Kröplin This Kröplin This KröplinStudy Study Study Count in 44 183 6 31 1 5 Group Average 35.87 Not 15.18Not 1.1 Not HPLC given given given Result Standard 8.541 — 4.210 — N/A —Deviation Median 35.30 40.7 15.550 15.6 1.1 <2 Value Lowest 19.60 — 9.70— 1.1 — Value in Group Highest 63.7 67 19.8 — 1.1 — Value in Group

The recommendations for setting cut-off values for the three genotypecategories are shown in Table 2:

TABLE 2 Cut-off Range (nmoles Genotype 6-mTGN/g Hb/hr) ClinicalRelevance Homozygote low <6.76 Patient should not receive 6-MP orAzathioprine due to risk of cytotoxicity by 6-TGN. Heterozygote6.76-23.60 Patient will safely tolerate low initial doses of 6-MP orAZA. Increasing doses should be accompanied by close monitor ofmetabolites. Wild type >23.60 Patient will safely tolerate standardinitial doses of 6-MP or AZA. Increasing doses should be accompanied byclose monitoring of metabolites

Cut-offs for the trimodal distribution determined for TPMT levelsassayed using perchloric acid precipitation and HPLC/fluorescencedetection are shown in comparison to the cut-offs for homozygote low,heterozygote and wild type genotypes recommended based on the MayoClinic radiochemical method. These two methods rely on different TPMTsubstrates, which may result in different enzyme kinetics and differentenzyme turnover rates. Both methods resulted in a trimodal distributionTPMT activity. Table 3 provides a correlation of the recommended rangesfor both methods:

TABLE 3 Prometheus Mayo Genotype (6-mTGN/g Hb/hr) (U/ml RBC) Homozygotelow <6.76 nmoles <5.0 Heterozygote 6.76-23.60 nmoles 5.0-13.7 Wildtype >23.60 nmoles 13.8-25.1

EXAMPLE III Survey of Acids for Protein Precipitation

This example describes the use of several different acids for theprecipitation of proteinaceous material from samples containing6-methylthioguanine.

A calibration standard curve and high controls were prepared asdescribed in Example I. Both the standard curve and controls contained6-methyl thioguanine (6-mTG), the product of the reaction catalyzed byTPMT, making the reaction portion of the protocol unnecessary. To thestandard curve and one set of tubes, 11 M perchloric acid (PCA) wasadded. In place of the perchloric acid, one of the following acids wasadded to each set of samples: 17 M acetic acid (glacial) to a finalconcentration of 2.83 M; 16 M nitric acid (HNO₃) to a finalconcentration of 2.67 M; 15 M phosphoric acid (H₃PO₄) to a finalconcentration of 2.5 M; 18 M sulfuric acid (H₂SO₄) to a finalconcentration of 3 M, or 6 M trichloroacetic acid (TCA) to a finalconcentration of 1 M. Samples were then processed and analyzed asdescribed in Example I.

As shown in Table 4, sulfuric acid, phosphoric acid and acetic acidtreatment of RBC lysate yielded TPMT activity determinations similar tothose obtained after perchloric acid precipitation. However,6-methylthioguanine was undetectable in lysates following treatment withtrichloroacetic acid or nitric acid. These results indicate thatsulfuric acid, phosphoric acid or glacial acetic acid can be used inplace of perchloric acid to preferentially precipitate proteinaceousmaterial from lysates without degrading a methylated purine product suchas 6-mTG in the supernatant.

TABLE 4 Mean TPMT activity Percent (nmol 6-mTG/gm Standard coefficientof Acid Hb) Deviation Variation PCA 62.829 0.63 1.01% H₂SO₄ 58.896 1.121.91% H₃PO₄ 58.937 0.42 0.71% Acetic 62.230 1.60 2.57% TCA 0.000 0.00 NAHNO₃ 0.000 0.00 NA

All journal article, reference, and patent citations provided above, inparentheses or otherwise, whether previously stated or not, areincorporated herein by reference.

Although the invention has been described with reference to the examplesabove, it should be understood that various modifications can be madewithout departing from the spirit of the invention. Accordingly, theinvention is limited only by the following claims.

We claim:
 1. A method of determining thiopurine methyltransferase (TPMT)activity in a subject, comprising the steps of: (a) reacting sampleobtained from said subject with a thiopurine derivative to produce amethylated purine product, provided that said thiopurine derivative isnot 6-mercaptopurine; (b) contacting said reacted sample with acid,thereby precipitating proteinaceous material from said reacted sample;(c) separating supernatant from said precipitated proteinaceousmaterial; and (d) detecting in said supernatant said methylated purineproduct, wherein the amount of said methylated purine product indicatesa level of thiopurine methyltransferase activity in said subject.
 2. Themethod of claim 1, wherein said subject is an inflammatory bowel diseasepatient.
 3. The method of claim 1, wherein said acid is perchloric acid.4. The method of claim 3, wherein said perchloric acid is 70% perchloricacid.
 5. The method of claim 1, wherein said thiopurine derivative is6-thioguanine.
 6. The method of claim 5, wherein said methylated purineproduct is detected by fluorescence.
 7. The method of claim 1, whereinsaid methylated purine product is detected by high performance liquidchromatography (HPLC).